In molecular biology, ligases act to create a phosphodiester bond between strands of DNA or RNA. DNA ligase connects broken DNA strands to form covalent phosphodiester bonds between 3′ hydroxyl ends of one nucleotide with the 5′ phosphate end of another, using ATP in the process. The known DNA ligases in mammalian cells include DNA ligase I, which ligates Okazaki fragments during lagging strand DNA replication. DNA ligase III complexes with DNA repair protein XRCC1 to aid in sealing base excision mutations and recombinant fragments, and DNA ligase II is an alternatively spliced form of DNA ligase III. DNA ligase IV complexes with XRCC4 and catalyzes the final step in the non-homologous end joining DNA double-strand break repair pathway. It is also required for V(D)J recombination, the process which generates diversity in immunoglobulin and T-cell receptor loci during immune system development.
DNA ligases have become an indispensable tool in modern molecular biology research for generating recombinant DNA sequences. For example, DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids. Common commercially available DNA ligases were originally discovered in bacteriophage T4, E. coli or other bacteria. Using these enzymes, the most efficient way for a ligase to link DNA fragments is to connect two sticky ends together from a restriction digest. The hydrogen bonds between the complementary bases aid ligase in holding the two ends together while the phosphodiester bond is formed.
DNA double-strand breaks (DSBs) occur at random upon genotoxic stresses and represent obligatory intermediates during physiological DNA rearrangement events such as the V(D)J recombination in the immune system. DSBs, which are among the most toxic DNA lesions, are repaired by either homologous recombination or nonhomologous end joining (NHEJ), preferentially by the nonhomologous end-joining (NHEJ) pathway in higher eukaryotes. Failure to properly repair DSBs results in genetic instability, developmental delay, and various forms of immunodeficiency. Repair of DNA double strand breaks (DSB) by the nonhomologous end-joining pathway in mammals requires at least seven proteins involved in a simplified two-step process: (i) recognition and synapsis of the DNA ends dependent on the DNA-dependent protein kinase (DNA-PK) formed by the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs in association with Artemis; (ii) ligation dependent on the DNA ligase IV.XRCC4.Cernunnos-XLF complex (Drouet et al. (2006) J Biol Chem. 281(38):27784-93).
It has been reported that T4 DNA ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. However, such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, and thus is insignificant in many molecular biological applications of T4 DNA ligase.
DNA ligase enzymes are of commercial interest for catalysis of a variety reactions. The present invention provides a useful complex that acts as a single-strand DNA ligase.